Interferon proteins have been classified into three types, alpha, beta and gamma based on antigenic and structural differences. Gamma interferon has a number of characteristics that differentiate it from alpha and beta interferons. Among these differences are antigenic distinctiveness and greater activity with regard to immunoregulation and antitumor effects. Human gamma interferon may be produced by T lymphocytes stimulated by mutagens or by antigens to which they are sensitized. It may also be obtained through cloning and expression techniques now well known to the art.
Gamma interferon containing cells, however obtained, are collected and are disrupted by various means such as osmotic shock, ultrasonic vibration, grinding or by high shear disruption. The disrupted cell-gamma interferon mixture is then processed to isolate the gamma interferon. The insoluble debris is separated by centrifugation and the gamma interferon containing supernatant is collected for purification. Several procedures are described in the art for separating and purifying gamma interferon from the supernatant collected from the centrifugation step.
European Patent Application No. 0,087,686 discloses a three step process for purifying human immune interferons from the cell free supernatant or extract from the crude interferon source. In the first step (for naturally occuring interferon), an affinity column, such as concanavalin-A Sepharose is used, followed by chromatography on a carboxymethyl silica column using an increasing salt gradient and finally, on a silica gel permeation column. If sufficient purity is not obtained, concentration and chromatography on either the TSK or CM column is used.
European Patent Application No. 0,063,482 discloses a purification process employing chromatographic methods using (1) Controlled Pore Glass (beads; 2) Concanavalin A-Sepharose; (3) Heparin-Sepharose or Procion Red-agarose; and (4) gel filtration.
European Patent Applications No. 0,107,498 and 0,077,670 disclose a purification scheme employing (1) polyethyleneimine precipitation; (2) pH precipitation of bacterial proteins; (3) concentration and dialysis; (4) chromatography on (a) carboxymethyl cellulose; (b) a calcium phosphate gel; (c) a carboxymethyl cellulose and (d) gel filtration resins.
These purification processes require a multitude of steps, have caused degradation of the interferon by degradation or aggregation of the interferon molecule, or otherwise result in a gamma interferon product obtained in low yield or with low activity.
It would be desirable to (1) provide a purification scheme to separate gamma interferon from the cell debris from the disrupted cells in which the gamma interferon was produced; (2) separate gamma interferon from cell contaminants in high yields and with high purity and activity; (3) separate recombinant gamma interferon from cell contaminants; and (4) separate gamma interferon from cell contaminants without substantially degrading the interferon. The purification process described below is such a process.